CN111450231B - Application of indirubin derivative in preparing synergist medicine of tacrolimus or cyclosporine - Google Patents
Application of indirubin derivative in preparing synergist medicine of tacrolimus or cyclosporine Download PDFInfo
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- CN111450231B CN111450231B CN202010089420.7A CN202010089420A CN111450231B CN 111450231 B CN111450231 B CN 111450231B CN 202010089420 A CN202010089420 A CN 202010089420A CN 111450231 B CN111450231 B CN 111450231B
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- tacrolimus
- phii
- cells
- indirubin derivative
- synergist
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Abstract
The invention discloses an application of indirubin derivative in preparing a synergist (or economizer) medicament of tacrolimus or cyclosporine, wherein the structural formula of the indirubin derivative is shown as formula 1,
Description
Technical Field
The invention relates to the field of pharmacy, in particular to application of indirubin derivatives in preparing a synergist drug of tacrolimus or cyclosporine.
Background
Indigo is the main component of indigo naturalis, and research proves that the indigo naturalis has a regulating effect on the expression of liver drug enzymes. Su Y et al extract from indigo blue the active ingredient thereof-indirubin [ Su Y, cheng X, tan Y H, et al Synthesis of a dual functional anti-MDR tumor agent PH II-7with elucidations of anti-tumor effects and mechanisms [ J ]. PLoS ONE,2012,7 (3): e32782]. PHII-7 is a derivative synthesized by using indirubin as a template, and has a structural formula of
It was found in earlier studies that it can regulate the expression of ABCB1 on cell surfaces, revealing the activation of ABCB1 by nuclear receptor proteins, which has been shown to be less toxic to normal cells at effective doses in vitro and in vivo experiments.
Tacrolimus (FK 506) is the most commonly used immunosuppressant in clinic at present, and is a first-line medicament for preventing and treating rejection reaction after immune system diseases such as nephrotic syndrome and organ transplantation due to strong immunosuppression effect and less adverse reaction. However, the drug has a narrow therapeutic window, large individual differences, and risks of complications such as diabetes affect the long-term survival of transplanted kidneys, and is expensive and needs to be taken continuously for a long time.
Tacrolimus is metabolized mainly by P450 oxidase (CYP 3A4 and CYP3A 5) in the liver and small intestine, and is a substrate of glycoprotein P-gp. The effect of the pentacster capsule on the concentration of tacrolimus in whole blood of heart transplant recipients [ J ]. Medical guide, 2017,36 (02): 158-162], the pentaester capsule has an effect on the pharmacokinetics of tacrolimus, and the combination with FK506 can obviously improve the FK506 blood concentration and bioavailability of volunteers and transplant recipients. Meanwhile, the liver function can be improved, the incidence rate of adverse reaction can be reduced, and the schisandrin contained in the five-ester capsule plays a role in inhibiting CYP3A activity and is also a P-gp inhibitor. While schisandrin A (Sch-A) is the main component in schisandrin lignans, which acts synergistically on tacrolimus by mediating the metabolism of CYP3A enzyme.
Although the five-ester capsule has a certain enhancement effect on the therapeutic effect of tacrolimus, the five-ester capsule is a traditional Chinese medicine composition, the components are complex, the action target point is not clear, and the small molecular compound has the advantages of simple structure, single component and convenience in accurately calculating the dosage in clinical application. Therefore, a small molecule compound needs to be found to be used as a synergist drug of tacrolimus.
Disclosure of Invention
The invention provides an application of indirubin derivatives in preparing a synergist drug of tacrolimus or cyclosporine, which aims at a small molecule synergist drug lacking tacrolimus or cyclosporine in the prior art.
In the specification, the code of the indirubin derivative is PHII-7.
The technical scheme provided by the invention is as follows:
the application of the indirubin derivative in preparing the synergist medicine of tacrolimus or cyclosporine is shown in the formula 1,
in 1Represents that NO can be carried out at any position on the benzene ring 2 And (3) substitution. Preferably, in one embodiment of the present invention, the indirubin derivative has the structural formula
Preferably, in an embodiment of the present invention, the potentiating agent is an agent that enhances the therapeutic effect of tacrolimus or cyclosporine.
More preferably, the above-mentioned therapeutic effect of increasing the tacrolimus or cyclosporine is to increase the peak concentration of tacrolimus or cyclosporine, the area under the drug time curve or the drug clearance rate.
In the embodiment of the invention, the inventor researches the influence of PHII-7 on the dynamics of Talcum Mo Siyao through animal experiments in rats, 8 rats are randomly divided into a control group and an experimental group, four rats are administrated by stomach infusion, and two groups of rats are subjected to blood sampling through jugular vein puncture of the rats at different time points before and after administration, FK506 blood concentration is measured, and main pharmacokinetic parameters of FK506 are calculated; detecting IC50 values of PHII-7 and schizandrin A (schisandra A, sch-A) on a human liver cancer cell strain HepG2 and a colon cancer cell strain LS174T by adopting an MTT method; detecting the influence of PHII-7 and Sch-A on the mRNA and protein expression level of PXR/CYP3A4/3A5/ABCB1 genes by using a real-time fluorescence quantitative PCR and immunoblotting method; constructing a double-luciferase reporter gene for stably transfecting the CYP3A5/ABCB1 promoter, and exploring the regulating effect of PHII-7 on the activity of the CYP3A5/ABCB1 promoter through PXR. The results showed that the area under the curve (AUC 0-t), the clearance of the drug (CL) was significantly increased in the experimental group compared to the control group (P)<0.05 A) is provided; the IC50 of PHII-7 against HepG2 and LS174T cells were (17.82.+ -. 1.33) and (7.17.+ -. 0.37) umol.L, respectively -1 IC50 of Sch-A for HepG2 and LS174T cells was (40.38.+ -. 4.1) and (44.1.+ -. 1.94) umol.L, respectively -1 The method comprises the steps of carrying out a first treatment on the surface of the After PHII-7 and Sch-A treatment of HepG2 and LS174T cells, the mRNA and protein expression of PXR, CYP3A4/3A5 and ABCB1 genes are obviously reduced, the dose dependence and the time dependence are presented, and the difference has statistical significance (P<0.05 A) is provided; warp yarnPHII-7 treatment transfected h-PXR cells reduced rifampicin-induced CYP3A5, ABCB1 expression and were concentration-dependent. PHII-7 can improve Cmax, AUC0-t and CL of FK506, which indicates that PHII-7 regulates metabolism and transport of FK506 through PXR-CYP3A5/ABCB1 signaling pathway, thereby increasing the curative effect of FK 506.
As a potentiator of the immunosuppressant tacrolimus or cyclosporine, the indirubin derivative (the structural formula of which is shown as formula 1) of the present invention has wide application in preparing auxiliary drugs in various situations, such as, but not limited to, preparing auxiliary drugs taken by patients after organ transplantation or hematopoietic stem cell transplantation, preparing auxiliary drugs for treating nephrotic syndrome, etc.
Patients usually need to take immunosuppressant after organ transplantation or hematopoietic stem cell transplantation, and the most commonly used immunosuppressant is tacrolimus or cyclosporine, so indirubin derivatives can be used as synergists of the immunosuppressant to assist in treatment of the immunosuppressant.
The indirubin derivative may be in the form of any suitable derivative, hydrate, pharmaceutically acceptable salt, or the like. Preferably, in an embodiment of the present invention, the indirubin derivative may be in the form of a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable acid addition salt thereof or a hydrobromide salt thereof.
The tacrolimus or cyclosporine potentiating agent may be in any suitable dosage form including, but not limited to, for example, tablets, capsules, oral formulations, microcapsules, injections or granules.
In another aspect of the invention there is provided a potentiator preparation of tacrolimus or cyclosporine comprising: indirubin derivatives represented by formula 1; other tacrolimus or cyclosporine potentiating agents.
In order to enhance the effect of indirubin derivatives, the indirubin derivatives can be combined with other tacrolimus or cyclosporine synergist medicines. Preferably, the synergist drug of the other tacrolimus or cyclosporine is a five-ester capsule.
The beneficial effects of the invention are as follows:
the invention systematically explains the synergistic effect of the indirubin derivative PH II-7 on tacrolimus and the molecular mechanism thereof for the first time: firstly, determining the influence of PH II-7 on the in vivo Talcum Mo Siyao pharmacokinetic parameters of rats through in vivo experiments, and further exploring the regulation effect of PH II-7 on the proteins such as CYP3A5/ABCB1 of the cells at the cellular level through primary liver and small intestine cells of rats, small intestine epithelial cell models, liver cell models, extracted liver microsomes and the like; finally, by constructing a CYP3A5/ABCB1 promoter double luciferase vector, a system mechanism of the PH II-7 on the effect and regulation of the PXR/CYP3A5/ABCB1 signal path network is explored, and a basis is provided for the development of new tacrolimus synergists in the future.
Compared with the five-ester capsule of tacrolimus synergist widely used in clinic at present, the indirubin derivative PH II-7 as a small molecular compound has the advantages of simple structure, single component, convenience in accurate dosage calculation in clinical application and better development prospect.
Drawings
FIG. 1 is a schematic diagram of a plasmid structure, wherein A is an h-PXR plasmid with cloning site of XhoI/KpnI, and B is a CYP3A5 plasmid with cloning site of KpnI/XhoI; c is the ABCB1 plasmid whose cloning site is KpnI/XhoI.
FIG. 2 shows the plasma concentration-time profile of FK506 in four groups of rats FK506, PHII-7, FK506+Sch-A and FK506+PHII-7
FIG. 3 is a graph showing the effect of PHII-7 and Sch-A on HepG2 and LS174T, with Sch-A as positive control, wherein (A) PHII-7 at different concentrations was applied to HepG2 cells for 24 hours, (B) Sch-A at different concentrations was applied to HepG2 cells for 24 hours, (C) PHII-7 at different concentrations was applied to LS174T cells for 24 hours, and (D) Sch-A at different concentrations was applied to LS174T cells for 24 hours;
FIG. 4 is a graph showing the effect of PHII-7 and schizandrin A on different genes in HepG2 and LS174T, wherein (A) PHII-7 was applied to HepG2 cells at different concentrations for 24 hours, (B) 18. Mu. Mol.L -1 PHII-7 of (C) different concentrations of Sch-A on HepG2 cells for 24 hours or 72 hours, (D) 40. Mu. Mol.L on HepG2 cells for 24 hours -1 Sch-A for 24 hours or 72 hours on HepG2 cells, (E) PHII-7 at various concentrations for 24 hours on LS174T cells, (F) 7μmol.L -1 PHII-7 of (C) was applied to LS174T cells for 24 hours or 48 hours, (G) Sch-A was applied to LS174T cells at various concentrations for 24 hours, (H) 44. Mu. Mol.L -1 Is applied to LS174T cells for 24 hours or 48 hours, P<0.05vs control group, ×p<0.01vs control;
FIG. 5 is a graph showing the effect of PHII-7 and schizandrin A on different proteins in HepG2 and LS174T, wherein (A) PHII-7 was applied to HepG2 cells at different concentrations for 24 hours, (B) 18. Mu. Mol.L -1 PHII-7 of (C) different concentrations of Sch-A on HepG2 cells for 24 hours or 72 hours, (D) 40. Mu. Mol.L on HepG2 cells for 24 hours -1 Sch-A for 24 hours or 72 hours on HepG2 cells, (E) PHII-7 at various concentrations for 24 hours on LS174T cells, (F) 7μmol.L -1 PHII-7 of (C) was applied to LS174T cells for 24 hours or 48 hours, (G) Sch-A was applied to LS174T cells at various concentrations for 24 hours, (H) 44. Mu. Mol.L -1 Is applied to LS174T cells for 24 hours or 48 hours;
FIG. 6 is a graphical representation of the transcriptional activity of the CYP3A5 or ABCB1 promoters in cells transfected with a reporter gene, wherein HepG2 cells have been transfected with a coding region vector encoding human PXR, simultaneously with GV238-CYP3A5-MCS-Luc reporter gene (A) or GV238-ABCB1-MCS-Luc reporter gene (B) vector, with or without PHII-7 (1, 9 and 18. Mu. Mol.L) -1 ) Or KTZ (25 and 50. Mu. Mol.L) -1 ) Blank control (0.1% DMSO) or RIF (10. Mu. Mol.L) -1 ) Treating for 24 hours; LS174T cells were transfected with the coding region vector encoding human PXR and simultaneously with the GV238-CYP3A5-MCS-Luc reporter (C) or GV238-ABCB1-MCS-Luc reporter (D) vectors, the cells were isolated with or without PHII-7 (1, 3.5 and 7μmol.L) -1 ) Or KTZ (25 and 50. Mu. Mol.L) -1 ) Blank control (0.1% DMSO) or RIF (10. Mu. Mol.L) -1 ) Treating for 24 hours; then detecting luciferase activity; the results show that indirubin derivatives can reduce the transcription activity of CYP3A5 or ABCB1 promoters induced by nuclear receptor PXR and weaken the transcription activity of CYP3A5 or ABCB1 promoters induced by rifampicinSex increase (concentration dependence, gradual decrease of promoter transcriptional activity with increasing indirubin derivative concentration),. P<0.05 indicates a statistically significant effect compared to the control group (n=3).
Detailed Description
The invention discloses application of indirubin derivatives in preparing a synergist drug of tacrolimus or cyclosporine, and a person skilled in the art can refer to the content of the indirubin derivatives and properly improve the technological parameters. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Experimental material and instrument
1. Experimental animal
SD rats, clean grade, male, quality (294.9 ±22.7) g, supplied by the common canvass laboratory animal company, animal use license number: SCXK 2016-0010.
2. Medicament
PHII-7 can be synthesized according to the invention patent application No. 94107957.0 or 200910068766.2. Dissolving the crude drug in DMSO to prepare 50 mmol.L -1 Stock solution, sch-A, is purchased from Chinese drug inspection institute, and the crude drug DMSO is dissolved to prepare 20 mmol.L -1 Storing the storage solution, subpackaging, and storing at-20deg.C for use, wherein the final concentration of DMSO in the dissolved drug solution<1%. FK506 was purchased from Shanghai Michelia Biochemical technology Co., ltd. And used as a raw material by dissolving 55% PEG400+25% propylene glycol +20% water to a desired solubility.
3. Culture medium
DMEM high sugar medium and fetal bovine serum were purchased from Gibco company.
4. Other reagents
MTT, verapamil, rifampin, ketoconazole were all purchased from Sigma; BCA quantification kit was purchased from the biotechnology company of nanking nuozan; dual Luciferase (LEF) assay reagents were purchased from Promega, USA; lipofectamine 2000 liposomes were purchased from ABM corporation; RNA extraction reagents were purchased from AxyGen company; both the reverse transcription kit and the fluorescent quantitative PCR (q-PCR) reagent were purchased from TaKaRa corporation, japan; plasmid extraction kit was purchased from beijing tiangen biochemical technology limited company; the rabbit anti-human beta-actin, ABCB1, CYP3A4 and CYP3A5 monoclonal antibodies are all purchased from abcam company; the murine anti-human PXR monoclonal antibody was purchased from Proteitech corporation; HRP-labeled goat anti-rabbit IgG antibodies and goat anti-mouse IgE antibodies were purchased from sequoyis, beijing; beta-actin, PXR, ABCB, CYP3A4 and CYP3A5 primers are synthesized by Huada gene company; the pcDNA-hPSR, ABCB1-MCS, CYP3A5-MCS luciferase reporter gene, renilla luciferase pRL-TK plasmid was purchased from Shanghai Ji Kai company.
5. Cell strain and cell culture
Both the human liver cancer cell line HepG2 and the colon cancer cell line LS174T were purchased from Shanghai Fuhe cell bank. In 10% foetal calf serum high sugar DMEM medium, 5% CO 2 Incubate at 37 ℃.
6. Experimental instrument
High performance liquid chromatographs (agilent, usa); QT-2A vortex mixer (Shanghai, product of qite analytical instruments limited); CO 2 Cell incubator (Heraeus, germany); inverted microscopes (Olypus, japan); microplate reader (clinic); q-PCR apparatus (applied biosystems, USA); electrophoresis apparatus (Burley Co., USA).
Example 1: in vivo pharmacokinetic experiments in rats
1. Grouping and handling of animals
The 8 rats were randomly divided into four groups, FK506 or PHII-7 single and combination groups: (FK506+Sch-A), or (FK506+PHII-7), four in each group, and administered by intragastric administration, rats were fasted for 8h (free drinking water)) FK506 1.89 mg.kg was administered to each group -1 、PHII-7 3mg·kg -1 、FK506+SchA：1.89mg·kg -1 +10mg·kg -1 Fk506+phii-7 was given to the experimental group: 1.89 mg/. Kg -1 +3mg·kg -1 Once administered, and 4 hours after administration, animal feed is administered. Rats of each group were pre-dose, post-dose 0.0167, 0.5, 0.75,
1. 2, 3, 4, 6, 8, 12 and 24 hours were collected by jugular puncture of rats, about 0.2mL was collected and placed in EDTA anticoagulant tube, and the blood samples were stored in a-80℃freezer prior to analysis.
2. Sample detection conditions
Chromatographic conditions: chromatographic column: accumore C18 (2.1X10 mm 2.6 μm), mobile phase: 0.1% formic acid+10 mM ammonium acetate aqueous solution (A): methanol (B), gradient elution, column temperature: 40 ℃, sample injection volume: 6. Mu.L.
Mass spectrometry conditions: AB SCIEX 5500 triple four-level rod tandem mass spectrometry system, wherein the ion source is ESI source, and air curtain air pressure is as follows: 35psi, impinging air: 8psi ionization voltage: 5500psi, ion source temperature: 550 ℃, spray gas: 55psi, auxiliary heater: 55psi, multiple Reaction Monitoring (MRM) mode. The detection conditions are shown in Table 1.
TABLE 1 sample detection conditions
3. Sample processing detection
mu.L of the plasma sample was placed in a 1.5mL centrifuge tube, and 180. Mu.L of an internal standard acetonitrile solution (TBTM 40 ng.mL) -1 ) Vortex 2min, centrifuge at 13000rpm (8 ℃) for 10min, take supernatant, sample 6. Mu.L for detection.
4. Experimental results
According to the results of the measurement of the blood concentration, the time is taken as an abscissa, the blood concentration is taken as an ordinate, FK506 medicine-time curves (shown in figure 2) of two groups of rats are drawn, and the calculation results of the FK506 medicine-time curves in the plasma of the rats are shown in table 2.
The results showed that FK506+PHII-7 group AUC compared to FK506+Sch-A group 0-t CL is obviousIncrease (P)<0.05 A) is provided; PHII-7 was suggested to enhance FK 506C like Sch-A max Increase AUC 0-t 、CL。
TABLE 2 pharmacokinetic parameters of FK506 in different groups of rats
* P <0.05vs fkh506+sch-a group.
Example 2: cell viability assay
HepG2 and LS174T cells in logarithmic growth phase were inoculated into 96-well plates (about 15000 cells per well) and cultured at 37℃with 5% CO 2 Culturing under the condition for 24 hours, adding medicines in groups, setting 3 parallel holes for each concentration, adding PHII-7 (0-30 umol.L) with different concentrations in experimental groups -1 ) And Sch-A (0 to 60 umol.L) -1 ) Adding an equal volume of culture medium into the negative control group, enabling the final volume of each hole to be 200uL, continuing to culture for 24 hours, adding 20uL of MTT into each hole, carrying out light-proof incubation for 4 hours, discarding the supernatant, adding 150uL of DMSO into each hole, carrying out shaking mixing, and measuring absorbance at the wavelength of 490nm of an enzyme-labeled instrument. The experiment was repeated three times to calculate the average half-maximal inhibitory concentration (50%concentration of inhibition,IC50 value).
Experimental results:
MTT colorimetric method was used to observe the cell growth inhibition. The results showed that PHII-7 and Sch-A had growth inhibitory effects on both HepG2 and LS174T cells, and that PHII-7 had IC 50's of (17.82.+ -. 1.33) and (7.17.+ -. 0.37) umol.L for both HepG2 and LS174T cells, respectively -1 . IC50 of Sch-A for HepG2 and LS174T cells was (40.38.+ -. 4.1) and (44.1.+ -. 1.94) umol.L, respectively -1 (the results are shown in FIG. 3).
Example 3: real-time fluorescent quantitative PCR (polymerase chain reaction) detection of PXR/CYP3A4/CYP3A5/ABCB1 gene expression
Inoculation of 2.5X10 5 HepG2 and LS174T cells in good condition at 2mL per well in 6 well plates, wherein the experimental group was treated as follows: 9 umol.L -1 PHII-7 incubation of HepG2 cells for 24h,18 umol.L -1 PHII-7 incubated HepG2 cells for 24h and 72h, respectively; 3.5 umol.L -1 PHII-7 incubation LS174T thinCytophyton 24h, 7umol.L -1 PHII-7 incubated LS174T cells for 24h and 48h, respectively; 20 umol.L -1 Sch-A incubate HepG2 cells for 24h,40 umol.L -1 Sch-A incubated HepG2 cells for 24h and 48h, respectively; 22 umol.L -1 LS174T cells were incubated for 24h,44 umol.L by Sch-A -1 LS174T cells were incubated for 24h and 48h respectively by Sch-A, control and treatment groups were collected respectively, total RNA was extracted from the cells using Trizol, cDNA synthesis was performed according to the instructions of the TaKaRa cDNA synthesis kit, and the expression level of each gene in the cells was detected by q-PCR with the reference beta-actin. Reaction conditions: pre-denaturation at 95℃for 30s, annealing at 95℃for 5s,60℃for 34s,40 cycles. Each group is provided with 3 parallel holes, after the reaction is finished, a threshold value is set, and the Ct value is input by software. Equation 2 according to the comparative Ct value method -ΔΔCt Where ΔΔct=experimental group (Ct Target gene -Ct Housekeeping genes ) Control group (Ct Target gene -Ct Housekeeping genes ) Obtaining the relative gene expression values among different groups.
Primers were designed using Primer 5.0 software based on Gene sequence information provided by the Gene-Bank database and synthesized by Huada genes company by BLAST analysis. The primer sequences used in this study are shown in Table 3.
TABLE 3 real-time fluorescent quantitative PCR primer sequences
Experimental results:
PHII-7 and Sch-A at different concentrations were applied to HepG2 and LS174T cells, respectively, and after different time periods of application, PXR, CYP3A4, CYP3A5 and ABCB1 gene mRNA expression was significantly reduced in HepG2 and LS174T cells after the drug administration treatment of PHII-7 and Sch-A compared with the control group, and was dose-dependent and time-dependent (results are shown in FIG. 4).
Example 4: immunoblotting method for detecting expression of PXR/CYP3A4/3A5/ABCB1 protein
Inoculation of 2.5X10 5 HepG2 and LS174T cells per mL were plated in 6-well plates at 2mL per well, with the experimental grouping being treated as follows: 9 umol.L -1 PHII-7 incubation of HepG2 cells 24h,18 umol.L -1 PHII-7 incubated HepG2 cells for 24h and 72h, respectively; 3.5 umol.L -1 PHII-7 incubated LS174T cells for 24h, 7umol.L -1 PHII-7 incubated LS174T cells for 24h and 48h, respectively; 20 umol.L -1 Sch-A incubate HepG2 cells for 24h,40 umol.L -1 Sch-A incubated HepG2 cells for 24h and 48h, respectively; 22 umol.L -1 Sch-A incubating LS174T cells for 24h,44 umol.L -1 LS174T cells are respectively incubated for 24h and 48h by Sch-A, cells of a control group and a treatment group are respectively collected, and protein is quantified after RIPA is lysed and used for Western blot detection. Performing SDS-polyacrylamide gel electrophoresis with a loading amount of 50ug per hole, transferring a protein sample to a PVDF membrane, sealing, and then respectively incubating PXR (1:1000), ABCB1 (1:1000), CYP3A4 (1:5000), CYP3A5 (1:10000) and beta-actin antibody (1:10000), incubating overnight at 4 ℃, incubating with a secondary antibody at room temperature for 1h, and performing chemiluminescence exposure.
Experimental results:
PHII-7 and schizandrin A with different concentrations are respectively acted on HepG2 and LS174T cells, after different time of action, compared with a control group, PXR, CYP3A4, CYP3A5 and ABCB1 protein expression of the HepG2 and LS174T cells is obviously reduced after the PHII-7 and schizandrin A are treated by drug addition, and the dosage dependence and the time dependence are shown in a result (shown in figure 5).
Example 5: transient transfection and reporter gene detection
And inoculating HepG2 or LS174T cells in the logarithmic growth phase into a 24-well plate, and when the fusion rate is 70-80%. Transfection was performed with Lipofectamine TM 2000 liposomes, phenol red free RPMI 1640. As internal controls pcDNA-hPRR or empty vector (455 ng), ABCB1-MCS or CYP3A5-MCS luciferase reporter (455 ng) and pRL-TK (90 ng) were used. After 6h transfection, 3 parallel wells were set up per group, cells were collected after 24h treatment with different drugs, washed 3 times with PBS, 100uL of cell lysate was added per well, lysed for 15min under light-shielding conditions, and luciferase activity was measured with an enzyme-labeled instrument. The relative luciferase activity measured after transfection of the cells by the plasmids tested was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity. Plasmids used in the experiments were supplied by Shanghai Ji Kai company (shown in FIG. 1), and the sequence information of the plasmids is shown in Table 4.
TABLE 4 PCR primers and PCR amplified product sizes
Experimental results:
the experiment studies PXR reactivations of ABCB1 and CYP3A5 promoters in reporter transfected HepG2 and LS174T cells by dual luciferase reporter gene experiments. As shown, in cells transfected with empty plasmids, the activity of the reporter gene was not affected by PHII-7 treatment. In hPrR transfected cells, PHII-7 treatment significantly activated the ABCB1 and CYP3A5 promoters, but when PHII-7 alone (HepG 2:1, 9, 18 umol.L -1 ；LS174T：1、3.5、7umol·L -1 ) Has no obvious effect on the activity of the promoter. While rifampicin (10 umol.L) -1 ) PHII-7 (HepG 2:1, 9, 18umol.L) was added during induction -1 ；LS174T：1、3.5、7umol·L -1 ) The ABCB1 and CYP3A5 promoters are significantly reduced in activity. KTZ was used as a positive control for ABCB1 and CYP3A5 inhibitors. These results indicate that PHII-7 is likely an antagonist of PXR (results are shown in FIG. 6).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The application of the indirubin derivative in preparing a synergist for improving the peak concentration, the area under a drug time curve or the drug clearance rate of tacrolimus, wherein the structural formula of the indirubin derivative is as follows:。
2. the use according to claim 1, wherein the indirubin derivative is in the form of a pharmaceutically acceptable salt thereof.
3. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is a tablet, capsule, microcapsule, injection or granule.
4. A potentiator preparation for increasing peak concentration, area under the drug time curve or drug clearance rate of tacrolimus, characterized in that said potentiator preparation comprises indirubin derivative and pentaester capsule; the structural formula of the indirubin derivative is shown as follows:。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1336602A1 (en) * | 2002-02-13 | 2003-08-20 | Giovanni Scaramuzzino | Nitrate prodrugs able to release nitric oxide in a controlled and selective way and their use for prevention and treatment of inflammatory, ischemic and proliferative diseases |
| CN101428067A (en) * | 2008-12-04 | 2009-05-13 | 中山大学 | Immunosuppressive agent and preparation method thereof |
| CN101573109A (en) * | 2006-12-28 | 2009-11-04 | 利默里克生物制药公司 | Methods and compositions for therapeutic treatment |
| CN102586191A (en) * | 2012-01-10 | 2012-07-18 | 浙江大学 | Construction and application of cell model for cotransfection of drug metabolic enzyme and transporter |
| CN102805867A (en) * | 2012-09-02 | 2012-12-05 | 江南大学 | Application of transcription factor NFATC3 as drug target in reversing multidrug resistance of tumor |
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2020
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1336602A1 (en) * | 2002-02-13 | 2003-08-20 | Giovanni Scaramuzzino | Nitrate prodrugs able to release nitric oxide in a controlled and selective way and their use for prevention and treatment of inflammatory, ischemic and proliferative diseases |
| CN101573109A (en) * | 2006-12-28 | 2009-11-04 | 利默里克生物制药公司 | Methods and compositions for therapeutic treatment |
| CN101428067A (en) * | 2008-12-04 | 2009-05-13 | 中山大学 | Immunosuppressive agent and preparation method thereof |
| CN102586191A (en) * | 2012-01-10 | 2012-07-18 | 浙江大学 | Construction and application of cell model for cotransfection of drug metabolic enzyme and transporter |
| CN102805867A (en) * | 2012-09-02 | 2012-12-05 | 江南大学 | Application of transcription factor NFATC3 as drug target in reversing multidrug resistance of tumor |
Non-Patent Citations (3)
| Title |
|---|
| CYP3A和MDR1基因多态性与他克莫司血药浓度的相关性;徐亚飞;《中国药物应用与监测》;第12卷(第1期);第59-62页 * |
| PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species;HongweiPeng;《European Journal of Pharmacology》;第718卷(第1-3期);Pages 459-468 * |
| P-糖蛋白和CYP3A酶介导的合并用药对他克莫司血药浓度的影响;王延飞;《中国药理学会第三届全国治疗药物监测学术年会资料汇编》;第19页 * |
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